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Histological analysis Untreated and decellularized samples were fixed in 4% formaldehyde and embedded in 3% agarose, dehydrated in an ascending series of alcohol, then embedded in paraffin and sectioned at 6 µm. Sections were stained with Gill’s Haematoxylin and Eosin (H&E, Merck), Safranin-O and Resorcin Fuchsin (RF, Klinipath, Duiven, the Netherlands). Additionally, collagen type II and elastin were immunohistochemically visualized. Antigen retrieval for the collagen type II antibody (II-II 6B3; DSHB, Iowa, USA) was achieved by incubating in 0.1% pronase in PBS for 30 minutes at 37°C. Antigen retrieval of elastin (BA-4) was carried out by incubation in 0.25% trypsin in PBS for 20 minutes at 37°C. 10% goat serum in PBS was used to block non-specific binding sites. Next, sections were stained for 1 hour with primary antibodies against collagen type II (1:100) or elastin (1:1000). An enzyme-streptavidin conjugate (HK- 321/325-UK; Biogenex, California, USA) in PBS/1% BSA at a dilution of 1:100 was used as label and visualized by Neu Fuchsin substrate (Chroma, Köngen, Germany). Cell-seeded cartilage scaffolds were immediately embedded in Tissue-Tek OCT Compound (Sakura, Alphen aan den Rijn, the Netherlands) after harvest, sectioned at 6 µm, fixed in acetone and stained with H&E. For SEM analysis, samples were dehydrated in a graded alcohol series, fractured by pulling at the distal end of the samples and dried with hexamethyldisilazane. Samples were then mounted on stubs, coated with palladium gold in a sputter coater (SC7620; Emitech/Quorum Technologies, Laughton, UK) and visually observed with a scanning electron microscope (JSM-6510; JEOL, Tokyo, Japan). Biomechanical testing Biomechanical properties of cartilage scaffolds were assessed using stress-relaxation- indentation as previously described [156]. In short, samples ( n =3 with 6 samples per donor) were placed in close-fitting stainless steel cylindrical wells of 5 mm in diameter, while immersed in PBS supplemented with antibiotic/antimycotic solution. Mechanical testing was performed with a materials testing machine (Zwick Z005, Ulm, Germany) equipped with a 10 N load cell, a built-in displacement control, and a cylindrical, plane ended, stainless steel indenter ( ∅ 0.35mm). A preload of 3 mN was first applied on the sample to locate the sample surface and measure sample thickness, and held for 5 minutes. Five consecutive strain steps in 5% increments were applied up to a maximum strain of 25%. Samples were then left to relax for 20 minutes at each step. A custom MATLAB ® script was used to convert the force- displacement data to stress-strain. Maximum stress (σ max ) equilibrium modulus (Eeq), relaxation time (τ) and relaxation half time (t ½ ) were determined from the stress-strain plots to determine intrinsic, flow-independent, and flow-dependent mechanical properties. [155] Scaffold cytocompatibility To assess toxicity and complete removal of the used chemicals during decellularization, the scaffolds were evaluated for their cytotoxicity with a methylthiazolyldiphenyltetrazolium bromide (MTT) assay. Bone-marrow derived mesenchymal stem cells (BMSCs) were isolated from bone marrow aspirates from patients undergoing total hip-replacement surgery (3 males, 67±5 Y), with informed consent and approval of the Medical Ethics Committee (Albert 134 CHAPTER 7